All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
Live-cell imaging has become a valuable technique for studying dynamic biological processes in real-time. The ability to visualize and track active processes in a single living cell has provided new insights into cellular architecture, membrane organization, dynamic protein assemblies, molecular organization, and cellular responses to external signals.
Central to these types of experiments is the knowledge of the general health of the culture and the cell of interest. While the morphological and biochemical changes that occur at different stages of apoptosis are well understood, unambiguously imaging these changes in living cells has been difficult. Several assays are available, which are aimed at detecting the specific biochemical changes that occur at the different stages of apoptosis, such as, phosphatidylserine exposure to the outer leaflet of the plasma membrane, mitochondrial dysfunction, activation of caspases, DNA fragmentation, and loss of membrane integrity. However, the current methods for these assays are generally disruptive to the cellular environment and, in most cases, are toxic to the cells.
A number of reasons make the detection of phosphatidylserine translocation to the extracellular face of the plasma membrane an attractive target for live-cell imaging. In healthy cells, plasma membrane asymmetry is closely regulated, and phosphatidylserine is restricted to the inner leaflet. Exposure of phosphatidylserine has been well established as a near universal indicator of apoptosis. In addition, phosphatidylserine provides abundant and easily accessible binding targets that can be detected without the need to penetrate into the cell. Moreover, it is an early event, thus monitoring phosphatidylserine exposure provides a way to observe the initiation of the apoptotic pathway before other changes are present. This is particularly useful for the detection of apoptotic processes in which progression into cell death does not occur; for example, in neuronal pruning or developmental axonal degeneration.
Annexins represent a highly conserved family of proteins that selectively bind to negatively charged, phosphatidylserine containing phospholipid membranes in the presence of calcium ions (Ca2+). Dying cells undergoing apoptosis expose these negatively charged lipids on the outer leaflet of the plasma membrane. Therefore, annexins selectively bind to apoptotic cells. This diagnostic application of annexins was first demonstrated using fluorescently labeled annexin A5 (Vermes et al. A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labeled Annexin V. (1995) J. IMMUNOL. METH. 184:39-51). The originally described annexin A5-based assay has been widely used in biological applications and has not been modified significantly. Modifications to the annexin A5-based assay have been limited to the use of different fluorophores that allow detection of fluorescent signals of different colors.
However, annexin-based probes described to date are impractical for live-cell imaging experiments since separate steps are required for binding of the fluorescent annexin probe to the apoptotic cells and subsequent removal of the unbound protein in order to reduce the background before analysis by fluorescence microscopy. Not only is the washing step an additional step, it places a limitation on real-time imaging of apoptotic cells and high-throughput screening for apoptotic cells. Thus, there exists a need in the art for a simpler apoptosis detection method, real-time and high-throughput apoptosis detection, as well as monitoring cell health.